Project Summary/Abstract The BCR-ABL fusion defines the most common molecular subtype of acute lymphoblastic leukemia (BCR-ABL+ ALL) in adults but historically conferred a poor prognosis. Incorporation of tyrosine kinase inhibitors (TKIs) that bind the ABL catalytic domain into frontline regimens can induce complete remissions in >90% of patients, but nearly all will relapse, typically with resistance mutations that disfavor drug binding. This suggests that relapses remain addicted to ABL kinase activity. In contrast, novel type IV inhibitors bind the ABL myristate site and allosterically modulate BCR-ABL function. We hypothesize that the combination of catalytic and allosteric TKIs can prevent the emergence of cross-resistant clones and cure a subset of treatment-nave BCR-ABL+ ALLs. We have established a diverse panel of 16 patient-derived xenograft (PDX) models of BCR-ABL+ ALL in which we can perform controlled and adequately powered pre-clinical trials to assess heterogeneity of response to in vivo combined blockade, develop predictive biomarkers, and establish models of acquired in vivo resistance. We have also developed an approach for characterizing therapeutic sensitivity in individual tumor cells that is amenable to minimal residual disease (MRD) specimens. Specifically, we use a microcantilever-based platform known as the suspended microchannel resonator (SMR) to assay drug sensitivity by measuring changes in the buoyant mass of individual cells exposed to targeted inhibitors ex vivo with femtogram-range sensitivity. These cells are then collected downstream of the SMR for single-cell RNA-Seq (scRNA-seq) to define transcriptional programs and cell states that modulate differential response to therapeutics. To test our hypothesis that combined ABL blockade has curative potential, and to meet the pressing need for rapid and robust approaches to characterize sensitivity within individual tumor specimens at MRD, we propose the following Specific Aims: (1) Test whether combined catalytic and allosteric BCR-ABL inhibition can cure some BCR-ABL+ ALL PDX models. Any models that relapse on combination therapy will be interrogated for mutational, transcriptional (both coding and non-coding RNA), and proteomic correlates of acquired in vivo resistance. (2) Define approaches to overcome therapeutic resistance directly within MRD in vivo. We will use the SMR to identify resistant subclones within MRD and apply scRNA-seq to define transcriptional programs and cell states mediating resistance for which bulk tumor assays would be insensitive. Defining patients who could be cured with combined ABL blockade would be transformative, just as combined PML-RAR targeting with arsenic and ATRA has proven to be in promyelocytic leukemias. The applicant Dr. Mark Murakami has outlined a 5-year career development plan to become an independent investigator in translational leukemia biology. He has assembled a distinguished group of mentors, advisors, and collaborators whose expertise spans the continuum of bench to bedside translation. The Dana-Farber Cancer Institute is the ideal environment for completion of his scientific and career goals given its outstanding research community and record of training independent physician-scientists.